Ultrasensitive electrochemiluminescence aptasensor for 8-hydroxy-2'-deoxyguanosine detection based on target-induced multi-DNA release and nicking enzyme amplification strategy

Abstract

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a principal stable marker of DNA oxidative damage. Sensitive and specific detection of 8-OH-dG is of great importance for early disease diagnosis. In this paper, we developed an electrochemiluminescence aptasensor for 8-OH-dG detection based on target induced multi-DNA release and nicking enzyme signaling amplification strategy. First, three kinds of short DNAs were aligned on the aptamers immobilized on the magnetic beads. In the presence of 8-OH-dG, the aptamer recognized and specifically bound with 8-OH-dG, leading to the release of three kinds of short DNAs and three-fold signal amplification. Then the released short DNAs hybridized with ferrocence (Fc) labeled hairpin DNA (Fc-HP) immobilized on the gold electrode to form a double strand DNA. Subsequently, nicking endonuclease (Nt.AlwI) recognized the asymmetric sequence in the dsDNA and cleaved the substrate strand (Fc-HP) into two parts, one fragments containing Fc would leave the surface of electrode. Based on the quenching effect of Fc on the electrochemiluminescence (ECL) of Ru(bpy)₃²⁺/TPA, a signal-on ECL aptasensor was developed. At the same time, three kinds of short DNAs were released again and reused to initiate the repeated cycles of hybridization-cleavage. Under double signal amplification, this aptasensor achieved a low detection of 25 fM and a wide linear range from 100 fM to 10 nM for 8-OH-dG. Besides, the amount of 8-OH-dG in urine samples derived from different people were determined with satisfactory results.

Keywords: 8-Hydroxy-2′-deoxyguanosine; Aptamer; Electrochemiluminescence; Signal amplification.